Preparation and evaluation of reference of enterohemorrhagic Escherichia coli strains and genomic DNA / 中国生物制品学杂志

@#Objective To screen enterohemorrhagic Escherichia coli(EHEC) strain and declare it as a standard strain of China Medical Bacterial Species Conservation and Management Center(CMCC).On the base,to prepare strain reference and genomic DNA reference of EHECand declare them as national drug reference with independent intellectual property rights in our country.Methods According to GB4789.6-2016 National Food Safety Standards-Food Microbiology TestDiarrheagenic Escherichia coil Test,the EHEC strain was screened from 160 Escherichia coli strains from patients with diarrhea and declared as a standard strain of CMCC according to management regulations.EHEC bacterial solution and genomic DNA solution were prepared,and freeze-drying technology was used to prepare 600 strain(10~3 CFU/sample) and genomic DNA(20 ng/sample) samples respectively.20 strain and 20 genomic DNA samples were randomly selected for uniformity test.Samples storing at 25 ℃ and 37 ℃ for 1,3,5 and 7 d were taken respectively to test the transportation stability.Then the samples were tested for the short-term storage stability by storing at 4 ℃ for 7,14 and 28 d,and for the long-term storage stability by storing at 20 ℃ for 14,28 and 60 d.Three laboratories were organized for collaborative calibration.20 food products were chosen as the substrate to evaluate the application effect of strain samples.Results The one EHEC strain selected from 160 Escherichia coli strains from patients was finally declared as the CMCC(B) 43207standard strain.In the uniformity test,F_(strainsample)=0.662 0.05,and eae,stxl and stx2 of 20genomic samples were all positive.After storage at 25 and 37 ℃ for 7 d,-20 ℃ for 60 d and 4 ℃ for 28 d,the viable bacteria content of the strain samples was still 103 CFU/sample,and eae,stxl and stx2 of the genomic samples were positive.EHEC strains and genomic DNA samples selected randomly were identified as EHEC by three laboratories,the viable bacteria content was 10~3 CFU/sample,and eae, stxl and stx2 were all positive.It was detected in 20 kinds of food substrates after adding samples,but not in the background control.EHEC strain and the genomic DNA sample were included in drug standard materials in our country,numbered 80024 and 80056,respectively.Conclusion The prepared EHEC strain and genomic DNA standard materials with independent intellectual property rights can improve the timeliness of EHEC testing and make up for the gap in our country.

Tiempo de excreción fecal de Escherichia coliproductora de toxina Shiga en niños con síndrome urémico hemolítico / Duration of fecal shedding of Shiga toxin-producing Escherichia coli among children with hemolytic uremic syndrome

Introducción. Conocer el tiempo de excreción fecal de Escherichia coli productora de toxina Shiga (Shiga toxin-producing Escherichia coli; STEC, por sus siglas en inglés) en pacientes con síndrome urémico hemolítico sería útil para controlar la transmisión de la enfermedad.Objetivos. 1) Analizar las características del tiempo de excreción de STEC. 2) Evaluar la asociación con las variables sexo, edad, necesidad de diálisis, antibióticos y serotipos de STEC.Población y métodos. Estudio prospectivo, observacional, longitudinal y analítico. Período 2013-2019. Se realizaron coprocultivos al ingresar y cada 5-7 días hasta obtener 2 negativos. Se definió tiempo de excreción desde el inicio de la diarrea hasta el primer negativo. Se confirmó STEC por detección de los genes stx1, stx2 y rfbO157 por reacción en cadena de la polimerasa. Se calculó la media (IC 95 %) y percentilos del tiempo de excreción de STEC, y se compararon las variables estudiadas mediante el test de t.Resultados. Se incluyeron 43 pacientes. La media de tiempo de excreción fue 10,2 días (IC 95 %: 8,92-11,59), rango: 3-22 días. El 90 % de los pacientes negativizaron el coprocultivo a los 15 días. No hubo diferencias según sexo (p = 0,419), edad (p = 0,937), necesidad de diálisis (p = 0,917), antibióticos (p = 0,147) ni serotipos (p = 0,231).Conclusión. El 90 % de los pacientes negativizó el coprocultivo a los 15 días del inicio de la diarrea, y todos, al día 22. No se encontró asociación entre el tiempo de excreción y las variables estudiadas.

Introduction. Knowing the duration of fecal shedding of Shiga toxin-producing Escherichia coli(STEC) among patients with hemolytic uremic syndrome would be useful to control disease transmission.Objectives. 1) To analyze the characteristics of STEC shedding duration. 2) To assess the association with sex, age, need of dialysis, antibiotics, and STEC serotypes.Population and methods. Prospective, observational, longitudinal, and analytical study in the 2013-2019 period. Stool cultures were done upon admission and every 5-7 days until 2 negative results were obtained. Shedding duration was defined as the period from diarrhea onset to the first negative result. STEC was confirmed with polymerase chain reaction detection of stx1, stx2, and rfbO157 genes. The mean (95 % CI) and percentile values of the STEC shedding duration were estimated, and the studied outcome measures were compared using the t test.Results. A total of 43 patients were included. The mean duration of shedding was 10.2 days (95 % CI: 8.92-11.59), range: 3-22 days. After 15 days, 90 % of patients had a negative stool culture. There were no differences in terms of sex (p = 0.419), age (p = 0.937), need of dialysis (p = 0.917), antibiotics (p = 0.147) or serotype (p = 0.231).Conclusion. Fifteen days after the onset of diarrhea, 90 % of patients had a negative stool culture, and all patients had one after 22 days. No association was observed between the duration of shedding and studied outcome measures.

Occurrence of Escherichia coli virulence genes in feces of wild birds from Central Italy

Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic, enterohemorrhagic and Shiga-toxins producing Escherichia coli (E. coli), enteropathogenic E. coli, enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular, eaeA encoding intimin, hlyA encoding for hemolysin, stx1 and stx2 genes encoding Shiga-toxins 1 and 2, respectively, were investigated. Results: Overall, 21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail, 12(9.91%) samples were positive for eaeA, 10(8.26%) for stx1, 4(3.31%) for hylA and 1(0.83%) for stx2. An owl (Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However, virulence genes characterizing EPEC, and EHEC strains were mainly found among seagulls, waterfowl and feral pigeons. Conclusions: Seagulls, waterfowl and feral pigeons, which frequently reach and contaminate rural, urban and peri-urban areas with their droppings, may be important sources of E. coli infection for other animals and humans.

A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic O157:H7 / 南方医科大学学报

OBJECTIVE@#To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7.@*METHODS@#The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains.@*RESULTS@#At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion.@*CONCLUSIONS@#We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.

An enterohaemorrhagic Escherichia coli outbreak spread through the environment at an institute for people with intellectual disabilities in Japan in 2005

Objective@#An enterohaemorrhagic Escherichia coli (EHEC) outbreak at an institute with multiple facilities for children and adults with intellectual disabilities was investigated to characterize the cases and identify risk factors for infection.@*Methods@#A case was defined as a resident, a staff member or a visitor at the institute from 16 May through 30 June 2005 testing positive for type 2 Vero toxin-producing EHEC O157:H7 (confirmed case) or exhibiting bloody diarrhoea for two or more days (probable case). We collected and analysed demographic, clinical, laboratory and individual behaviour data to identify possible risk factors for infection and infection routes.@*Results@#We recorded 58 confirmed cases, of which 13 were symptomatic. One probable case was also found. The median age of the patients was 37 years (range: 6–59 years). Thirty-six patients (61%) were male. Thirteen patients (93%) had diarrhoea and six (43%) had abdominal pain. Two developed haemolytic-uraemic syndrome but recovered. All the patients were treated with antibiotics and tested negative after treatment. Some residents had problems with personal hygiene. The residents of one of the facilities who cleaned a particular restroom had 18.0 times higher odds of being infected with EHEC (95% confidence interval: 4.0–102.4) than those who did not.@*Discussion@#The source of the outbreak could not be identified; however, the infection may have spread through environmental sources contaminated with EHEC. We recommend that institutional settings, particularly those that accommodate people with intellectual disabilities, clean restrooms as often as possible to reduce possible infection from contact with infected surfaces.

Occurrence of Escherichia coli virulence genes in feces of wild birds from Central Italy

Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic, enterohemorrhagic and Shiga-toxins producing Escherichia coli (E. coli), enteropathogenic E. coli, enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular, eaeA encoding intimin, hlyA encoding for hemolysin, stx1 and stx2 genes encoding Shiga-toxins 1 and 2, respectively, were investigated. Results: Overall, 21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail, 12(9.91%) samples were positive for eaeA, 10(8.26%) for stx1, 4(3.31%) for hylA and 1(0.83%) for stx2. An owl (Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However, virulence genes characterizing EPEC, and EHEC strains were mainly found among seagulls, waterfowl and feral pigeons. Conclusions: Seagulls, waterfowl and feral pigeons, which frequently reach and contaminate rural, urban and peri-urban areas with their droppings, may be important sources of E. coli infection for other animals and humans.

Development and evaluation of loop-mediated isothermal amplification assay for the rapid detection of Escherichia coli and its microbial toxin / 中国医师杂志

Objective To establish and optimize a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Escherichia coli and its microbial toxin.Methods The LAMP reaction system and reaction conditions were determined by optimizing LAMP reaction,and the optimized LAMP system was used for the detection.Results Primers targeting shiga toxin (stx) gene and O157 antigen gene rfbe were designed.The established and optimized LAMP amplification system contained 1.2 mmol/L dNTPs,10 mmol/L MgSO4,0.4 mol/L betaine,1 μl 10 × Bst DNA polymerase Buffer,8 U Bst DNA polymerase fragment,2 μl DNA template,and the ratio of inner-primer (FIP and BIP) and outerprimer (F3 and B3) were 8∶ 1.Time and temperature for LAMP was 60 min,60 ℃.The sensitivity was 103 times higher than polymerase chain reaction (PCR),reached 5 × 101 CFU/ml.When LAMP was applied to 19 reference strains,102 EHEC strains,the specification was 100％ while identification rate of rfbe,stx1 and stx2 gene reached 100％,95.2％,92.9％.Conclusions The LAMP method showed a promising prospect for the rapid detection of common nosocomial pathogens microbial toxin.

Comparative study on the epidemiological aspects of enterohemorrhagic Escherichia coli infections between Korea and Japan, 2006 to 2010

BACKGROUND/AIMS: To compare the epidemiological aspects of enterohemorrhagic Escherichia coli (EHEC) between Korea and Japan by analyzing the current state of EHEC infection outbreaks and related risk factors. METHODS: We investigated the epidemiological aspects of EHEC infection cases between Korea and Japan from 2006 to 2010. The following factors were analyzed: national prevalence rate (PR), regional prevalence rate, epidemic aspects (i.e., Cases related to gender), male to female morbidity ratio, age, and seasonal distribution. RESULTS: In total, there were 254 cases of EHEC with an average PR of 0.11 per 100,000 populations in Korea from 2006 to 2010. During the same period in Japan, there were 20,883 cases of EHEC with an average PR of 3.26 per 100,000 populations. The PR in Japan was significantly higher than that in Korea (p 50%) observed for individuals younger than 9 years. EHEC is an emerging zoonosis and may be caused by consumption of raw or undercooked meat products from ruminants. CONCLUSIONS: This study provides a quantitative analysis of the epidemiological aspects and risk factors of EHEC infections in Korea and Japan and will provide insight on effective future strategies to reduce these infections.

Enteropatógenos de importancia en salud pública / Relevant public health enteropathogens

La diarrea continúa siendo la tercera causa de muerte en niños menores de 5 años, pese a los avances recientes en el manejo y prevención de esta enfermedad. Es causada por múltiples patógenos, sin embargo, la prevalencia de cada uno varía según el grupo de edad, la zona geográfica y el escenario donde se registran los casos (comunitario vs hospitalario). Los patógenos de mayor relevancia en salud pública son aquellos asociados con mayor carga de enfermedad, severidad, complicaciones y mortalidad. En nuestro medio, el norovirus, Campylobacter y las E. coli diarrogénicas, son los patógenos más prevalentes a nivel comunitario en niños. En este artículo se revisa la epidemiología local y las potenciales áreas de desarrollo en cinco patógenos seleccionados: rotavirus, norovirus, E. coli productora de toxina Shiga (STEC), Shigella y Salmonella. De estos, el rotavirus es el más importante en la población pediátrica y el principal responsable de la mortalidad infantil por diarrea. La introducción de la vacunación contra rotavirus en nuestro país tendrá un importante impacto en la carga de enfermedad y la mortalidad por diarrea. Sin embargo, se requieren estudios de vigilancia para determinar el impacto de la vacunación y el cambio en la epidemiología de la diarrea en el Perú luego de la introducción de nuevas vacunas, así como la vigilancia de las tasas de resistencia antibiótica para las bacterias de importancia clínica.

Diarrhea remains the third leading cause of death in children under five years, despite recent advances in the management and prevention of this disease. It is caused by multiple pathogens, however, the prevalence of each varies by age group, geographical area and the scenario where cases (community vs hospital) are recorded. The most relevant pathogens in public health are those associated with the highest burden of disease, severity, complications and mortality. In our country, norovirus, Campylobacter and diarrheagenic E. coli are the most prevalent pathogens at the community level in children. In this paper we review the local epidemiology and potential areas of development in five selected pathogens: rotavirus, norovirus, Shiga toxin-producing E. coli (STEC), Shigella and Salmonella. Of these, rotavirus is the most important in the pediatric population and the main agent responsible for child mortality from diarrhea. The introduction of rotavirus vaccination in Peru will have a significant impact on disease burden and mortality from diarrhea. However, surveillance studies are needed to determine the impact of vaccination and changes in the epidemiology of diarrhea in Peru following the introduction of new vaccines, as well as antibiotic resistance surveillance of clinical relevant bacteria.

Construction and preliminary study of biological functions of O157∶H7 typeⅢsecre-tion system effector NleF gene knockout mutant / 军事医学

Objective To construct Escherichia coli O157∶H7 T3SS effector NleF gene knockout mutant and its com-plementary strain, and probe its effects on bacterial growth and cell death .Methods T3SS Effector NleF gene knockout mutant ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 was measured every hour , and the growth curve was drawn .HeLa cells were infected with three kinds of strains , the supernatant of LDH release was detected with cytotoxicity detection kit ,and the cytotoxicity was calculated .Results ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/NleF infected cells.Conclusion EHEC O157∶H7 T3SS Effector NleF has no significant effect on bacterial growth ,but might inhibit host cell death caused by bacterial infection .

Prokaryotic expression, purification, identification, and polyclonal antibody prepa-ration of enterohemorrhagic Escherichia coli effector NleB1 / 军事医学

Objective To construct a prokaryotic plasmid expressing the recombinant protein of enterohemorrhagic Escherichia coli(EHEC) effector NleB1 and to prepare the polyclonal antibody of mouse anti-NleB1.Methods The nleB1 (990 bp) gene was amplified from the genome EHEC O157∶H7 and cloned into the expression plasmid pET24a to construct the recombinant plasmid pET24a-nleB1 that was transformed into E.coli BL21(DE3).After induction with isopropylthio-gelactoside( IPTG) , the His-tag fusion proteins were purified by Ni+affinity chromatography and gel slices.The polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant proteins and analyzed by Western blotting and ELISA.Results The pET24a-nleB1 recombinant plasmid was successfully constructed, the fusion protein was ex-pressed and purified,and the polyclonal antibody was obtained by immunizing mice with purified fusion protein.Western blotting and ELISA staining demonstrated that the polyclonal antibody was successfully obtained.Conclusion The prepara-tion of the polyclonal antibody against EHEC O157∶H7 NleB1 will be of help for further studies on the function of NleB1 protein.

Molecular characterization of Escherichia coli O157:H7 strains isolated from different sources and geographic regions

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.

Awareness of Major Zoonoses among Dairy Farmers in Gyeonggi Province / 農村醫學 地域保健

OBJECTIVES: We surveyed awareness levels of brucellosis, Q fever and enterohemorrhagic Escherichia coli (EHEC) among dairy farmers in Gyeonggi Province to suggest directions for public education and public relations. METHODS: We designed questionnaires to evaluate awareness of 3 major zooneses. We conducted a questionnaire survey to assess knowledge of the general characteristics of them, information sources for the awareness of zooneses, and the mode of transmission. Subjects were 716 workers from 482 dairy farms in Gyeonggi province. RESULTS: The awareness levels for brucellosis, Q fever, and EHEC were 90.2%, 2.5% and 56.6%, respectively. Awareness of brucellosis and EHEC were tended to increase with higher number of school years. Television was the most common route of information for these zoonoses. Most common responses for questions concerning the method of transmission for each zoonoses, 'Contact with parturient fluid or placenta of animal' was 63.2% for brucellosis, 'Ingestion of raw meat or residual product' was 66.7% and 64.2% for Q fever and EHEC, respectively. The most common reason why dairy farmers think that it is difficult to prevent zoonoses was the inconvenience of wearing protection. CONCLUSIONS: Education programs for zoonoses, especially Q fever, are needed for dairy farmers. In addition, publicity information activities about prevention of zoonoses are needed for high risk groups, such as the dairy farmers surveyed.

An outbreak of inapparent non-O157 enterohemorrhagic escherichia coli infection / 대한내과학회지

No abstract available.

Intrafamilial Spread of Diarrhea-associated Hemolytic Uremic Syndrome

Diarrhea-associated hemolytic uremic syndrome(D+ HUS) is induced by enterohemorrhagic Escherichia coli(EHEC) and is characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The disease is usually transmitted by meat and water contaminated by excreta of domestic animals. We report a son and his mother with diarrhea-associated hemolytic uremic syndrome that spread within the family.

Efecto citotóxico en colon humano de Escherichia coli enterohemorrágico aislado de terneros con diarrea sanguinolenta / Cytotoxic effect in human colon of enterohemorrhagic Escherichia coli isolated from calves with bloody diarrhea

Escherichia coli productor de toxina Shiga (STEC) es el patógeno emergente en alimentos de mayor impacto, siendo su principal reservorio el ganado bovino. STEC puede causar diarrea, colitis hemorrágica y síndrome urémico hemolítico. El presente trabajo estudió la acción citotóxica de dos cepas de STEC aisladas de heces de terneros diarreicos en colon humano in vitro. Los fragmentos se montaron como un diafragma en una cámara de Ussing y se incubaron con las cepas patógenas. El flujo neto absortivo de agua (Jw) disminuyó y la corriente de cortocircuito (Isc) aumentó significativamente (P < 0,01) con respecto al control negativo. Los tejidos presentaron erosión de la mucosa, exfoliación del epitelio, y presencia de pseudomembranas en el lumen. A nivel de la lámina propia se observaron lesiones circulatorias leves. Una moderada infiltración de neutrófilos se observó en el lumen y en las células epiteliales. Las criptas colónicas no se vieron afectadas. El grado de lesión fue similar en ambas cepas experimentales. Este es el primer estudio que demuestra que cultivos de cepas de STEC aisladas de ganado bovino producen efectos citotóxicos en colon humano in vitro.

Shiga toxin-producing E. coli (STEC) is one of the most important emergent pathogen in foods, being its main reservoir bovine cattle. STEC can cause diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. The present work have studied the cytotoxic action in human colon of cultures of two STEC strains isolated from faeces of calves with bloody diarrhea. Colonic mucosa was mounted as a diaphragm in a Ussing chamber and incubated with the cultures of pathogenic strains. Net water flow (Jw) decreased and the short-circuit current (Isc) increased significantly (p < 0,01) compared to negative control. Tissues showed an erosion of the mucose, epithelial exfoliation, and presence of pseudo-membranes in the lumen. Mild circulatory lesions were observed in the lamina propia. A moderate neutrophils infiltration was observed in the lumen and into the epithelial cells. Colonic crypts were not disrupted. Both experimental strains caused a similar lesion on colon tissues. This is the first study that shows that cultures of STEC strains isolated from bovine cattle produce cytotoxic effects in vitro in human colon.

Clinical Significance of Escherichia coli O26 Isolated from Clinical Specimens / 대한임상미생물학회지

BACKGROUND: Non-O157 human isolates among enterohemorrhagic Escherichia coli (EHEC) serogroup have been reported with increasing frequency in recent years; the serotype O26 is the most common among the non-O157 isolates. We performed serotyping of E. coli isolates with O157, O26, and O111 antisera at Ulsan University Hospital and identified 27 isolates of O26. The purpose of this study was to investigate the clinical significance of E. coli O26 isolates. METHODS: During the 24-month period from January 2002 to December 2003, E. coli isolates were serotyped when requested by the physician because of bloody diarrhea or when blood was noted in the stool specimen at the laboratory. The isolates were identified biochemically by Vitek 1 (BioMerieux Vitek Inc., Mo., USA) and serotyped using diagnostic antisera of O157, O26, and O111 (NIH, Korea). When a positive agglutination reaction was shown, the patient's was reviewed retrospectively. RESULTS: Of 4,921 isolates of E. coli during the 2-year period, 200 isolates were serotyped and 27 (13.5%) were identified as serotype O26. These were isolated from stool (13 isolates), urine (9), pus (1), blood (1), and bile (1). Among the 13 patients whose stool specimens grew E. coli O26, 12 had watery diarrhea and 7 bloody diarrhea; two patients had thrombocytopenia and purpura simultaneously. Two patients with watey diarrhea, two with bloody diarrhea, and one with TTP were among the 7 patients with E. coli O26 in the urine. Finally, one patient each with blood isolate and bile isolate of E. coli O26 both had acute gastroenteritis. CONCLUSIONS: Most of the patients infected with E. coli O26 had clinical manifestations consistent with EHEC infections. E. coli isolates from patients with boody diarrhea should be serotyped with O157 and O26 antisera.